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ABSTRACT The protein α-syn adopts a wide variety of conformations including an intrinsically disordered monomeric form and an α-helical rich membrane-associated form that is thought to play an important role in cellular membrane processes. However, despite the high affinity of α-syn for membranes, evidence that the α-helical form of α-syn is adopted inside cells has thus far been indirect. In cell DNP-assisted solid state NMR on frozen samples has the potential to report directly on the entire conformational ensemble. Moreover, because the DNP polarization agent can be dispersed both homogenously and inhomogenously throughout the cellular biomass, in cell DNP-assisted solid state NMR experiments can report either quantitatively upon the structural ensemble or can preferentially report upon the structural ensemble with a spatial bias. Using DNP-assisted MAS NMR we establish that the spectra of purified α-syn in the membrane-associated and intrinsically disordered forms have distinguishable spectra. When the polarization agent is introduced into cells by electroporation and dispersed homogenously, a minority of the α-syn inside HEK293 cells adopts a highly α-helical rich conformation. Alteration of the spatial distribution of the polarization agent preferentially enhances the signal from molecules nearer to the cellular periphery, thus the α-helical rich population is preferentially adopted toward the cellular periphery. This demonstrates how selectively altering the spatial distribution of the DNP polarization agent can be a powerful tool for preferential reporting on specific structural ensembles, paving the way for more nuanced investigations into the conformations that proteins adopt in different areas of the cell. 

Magic‐angle spinning (MAS) NMR coupled with dynamic nuclear polarization (DNP) has the possibility to increase the sensitivity of MAS NMR by several orders of magnitude. While DNP enables many experiments that are sensitivity limited, such as those on dilute samples or those that measure long‐range distances, interpretation of DNP NMR spectra is often limited by broad lines and chemical shift degeneracy. Segmental isotopic labeling using split intein technology can provide an opportunity to overcome this issue. Isotopic labeling of only a segment of a protein that is otherwise unlabeled reduces the chemical shift degeneracy. In this article, we describe the current state of the art for producing segmentally isotopically labeled proteins using split inteins. We discuss some of the potential applications of segmental isotopic labeling, particularly those that exploit the increased experimental sensitivity of DNP‐enhanced MAS NMR spectroscopy